Device for in situ analysis and/or treatment consisting of a flexible rod and micro system fixed at one end of said flexible rod

ABSTRACT

This invention concerns an apparatus for chemical or biological analysis or treatment in situ comprising (i) a microsystem for investigation of a substrate and/or for delivery of active agents in a substrate and (ii) a flexible rod to one end of which the microsystem is attached and the other end of which is intended for the control of said microsystem. The microsystem is advantageously of the type comprising a support on the surface of which predefined regions are arrayed, each containing different chemical or biological substances for investigation or treatment of the substrate where the microsystem is brought in contact thanks to the flexible rod.

This application claims the benefit of the filing date of U.S.application Ser. No. 60/246,571 filed Nov. 8, 2000.

This invention concerns an apparatus making it possible to carry out byremote control an investigation and/or treatment in situ in a substrate,for example, of tissues or organs, consisting of a flexible rod, to oneend of which an investigation and/or treatment microsystem is attached.

Investigation microsystems employing an array of biological moleculesplaced in given positions on a surface are described in the prior art.Those systems, known as “biochips” or DNA chips, are useful for theinvestigation of polynucleotide or amino acid sequences. Examples ofsuch systems are described, for example, in the European patentapplications published under No. 619,321, No. 373,203 and No. 691,978.Other investigation microsystems are, for example, the microanalysistests using ligand/receptor type reactions or microimmunoanalyses usingantigen/antibody type reactions.

Apparatuses for in vivo investigation of organs or tissues are alsoknown in the prior art, such as a catheter consisting of a flexible tubeinserted in the vessels or through the natural routes, associated, forexample, with a laser, an optical fiber, a probe or a sensor. Thoseapparatuses can also make possible the administration of activesubstances like drugs or diagnostic agents, such as, for example, theapparatus described in U.S. Pat. No. 5,938,595.

The inventors have now designed a remote-controlled apparatus foranalysis and/or treatment in situ combining the two technologiesmentioned above. It appears useful, in fact, to have available new meansof investigation and/or treatment in situ in a substrate of an organismin vivo or in vitro the least traumatizing as possible, particularly inthe case of a human patient, and which makes it possible to access (i)information useful for diagnosis as well as for screening, for example,for therapeutic indications, or (ii) new methods of administration ofactive pharmaceutical agents directly in a substrate consisting, forexample, of target cells.

This objective is attained according to the present invention thanks toan apparatus for chemical or biological analysis or treatment in situcomprising (i) a microsystem for investigation of a substrate and/or fordelivery of active agents in a substrate and (ii) a flexible rod to oneend of which the microsystem is attached and the other end of which isintended for the control of said microsystem, and in that, in the caseof an investigation, the microsystem is not of the type based onanalysis of the emission and detection of a fluorescent signal.

Said microsystem and said flexible rod are advantageously kept togetherin situ. Thus, according to the invention, said microsystem and saidflexible rod remain joined to one another upon use in situ and areseparated only before or after the operation in situ, so that themicrosystem does not constitute a system implantable or releasable insitu.

In a first embodiment, the microsystem is of the type comprising asupport on the surface of which predefined regions are arrayed, eachcontaining different chemical or biological substances for investigationor treatment of the substrate into which the microsystem is brought incontact thanks to the flexible rod. Said surface contains at least twoand preferably more than 100 and especially preferably more than 1000predefined regions on a surface in the order of several cm² andpreferably in the order of 1 cm² or less. Each predefined area containsa different chemical or biological substance, but the process accordingto the invention also allows several or even all of said predefinedregions to contain the same chemical or biological substance, forexample, in case of the same analysis or of delivery of the same activesubstance in time.

The support can contain several faces, at least one of which consists ofan active surface. The latter is the site of one or more biologicalbonding agents. In the midst of said active surface predefined regionsare arrayed.

This surface of approximately one cm² can be flat and is situated in asingle plane, and it can also be ribbon-shaped and spiral-wound on arigid support which extends the flexible rod and then presents themicrosystem of the same nature as the previous one, but of a moreelaborate configuration. FIG. 1 represents a working example of anapparatus according to the invention, consisting of a flexible rod (1)like, for example, a deformable catheter, at the end of which themicrosystem (2) is inserted in the opening of the flexible rod, themicrosystem consisting of a support (3) on which a ribbon (4) is woundor specific antibiodies of an antigen present in the substrate analyzedare fixed. The support (3) is rigid, while the flexible rod (1) isrelatively deformable. The flexible rod (1) can be combined with anendovascular exploration system (5) like, for example, an endoscope.

The apparatus of the invention can be combined with an endocavitary toendovascular exploration system making it possible to bring themicrosystem in contact with or in proximity to the target substrate.

In a second embodiment, the apparatus of the invention can consist of aflexible rod, possibly combined with an endocavitary or endovascularexploration system. The latter ends in a microsystem consisting of anarticulated segment composed of an alternation of rigid substances(platinum ball type) and of more or less hydrophilic gel (hydrogel) onwhich the reactive groups, molecules or substances are fixed. The activelayer surrounding the rigid support can be a flat ribbon or a round cordon which the reagents are deposited and fixed. It can involve ligandsand, notably, antigens or antibodies, but also nucleotides. Thus, inthis embodiment the microsystem consists of a support coming in the formof a ribbon wound around one end of the flexible rod. An example of thisembodiment is represented on FIG. 1 of the attached drawings.

As previously indicated, each predefined area can contain the sameactive chemical or biological substance, for example, in case of thesame analysis or of delivery of the same active substance reproduced intime. Each predefined area can also contain a different chemical orbiological substance, and the microsystem then supporting a plurality ofdifferent chemical or biological substances is intended to carry out amultiple analysis.

The apparatus according to the invention is useful in:

the therapeutic field for in situ delivery of agents active in asubstrate, and

the field of diagnosis for in situ analysis of a substrate.

In particular, the apparatus of the invention is useful for therapeuticapplications conducted within the framework of guided therapy, asproposed for intracardiac gene therapy aimed at bringing autologousmyeloid cells in situ after having evaluated with the endocavitary probethe state of the cell tissue of the myocardial area to be treated. Thisis with the aid of different systems and, in particular, of themicrosystems of diagnosis or therapeutic follow-up of the invention.

Within the scope of the therapeutic applications, the biological and/orchemical substances arrayed in each predefined region of the surface ofthe microsystem are active agents, notably therapeutic substances. Theactive agents can be any substance useful in the treatment of diseasesrequiring an intervention in target cells or tissues, like cancer cells,centers of infection, etc.

Within the scope of the diagnostic applications, the biological and/orchemical substances arrayed in each predefined region of the surface ofthe microsystem are substances making it possible to detect specificanalytes of the substrate where the microsystem is taken, or evenradio-labeled substances making possible the remote display of the organtargeted by any known medical imaging technique.

Said substances can be fixed in each predefined region of the supporteither reversibly, in order possibly to be released again by themicrosystem so that they may perform their function “in vivo”, afterremoval of said microsystem, or irreversibly on said surface. Fixationcan be carried out by simple adsorption or by means of a couplingproduct.

The active agents are released in the substrate:

by simple contact with the latter, in which case they are physicallyprotected, as described below, until the microsystem is placed in saidsubstrate, or

thanks to a complementary system of release in the microsystem.

A preferred method of use of the therapeutic application of theapparatus of the invention consists in utilizing active substances whichare nucleic acids, like naked DNAs or antisense oligonucleotides, fixedon the surface of the support of the microsystem by hybridization. Butthe active substances can also be proteins or antibodies fixed on eachpredefined region thanks to an immunological bond. By way of example ofa microsystem forming part of a treatment apparatus according to theinvention, one can mention the bionic chips which electrically controlcell activity, so as to release therapeutically active agents.

For the applications of diagnosis and, more widely, of analysis of asubstrate, the microsystem of the apparatus of the invention is aninvestigation microsystem.

According to a preferred embodiment of the apparatus of the invention,the chemical or biological substances are capable of reacting withcorresponding substances possibly present in the substrate where theinvestigation microsystem is brought thanks to the flexible rod.

Thus, the microsystem is preferably a receptor/ligand microsystem usingpairs of chemical or biological substances in which either or both ofthe members of the pair are fixed on each predefined region, notably fortherapeutic applications.

The microsystem according to the invention advantageously usesbiological substances which are polynucleotide or amino acid sequences.The invention envisages as chemical or biological substances present ineach predefined region chemical or biological substances constituting:

an array of polynucleotide sequences capable of being hybridized withnucleic acids present in the substrate notably constituting theinvestigation site, or

an array of peptides, polypeptides or proteins capable of reacting witha corresponding receptor or immunologically with antibodies or antigenspresent in the substrate notably constituting the investigation site.

But the investigation microsystem can also use chemical substancescapable of reacting according to varied chemical reactions withcorresponding chemical substances present in the substrate analyzed.Thus, the investigation microsystem according to the invention makespossible the simultaneous analysis of several physiological factors insitu.

Taking into account the diversity of types of biological or chemicalreactions capable of being used in the investigation microsystem, theapparatus of the invention makes possible the identification of genes ortheir components of DNA, of RNA, of pertinent nucleotide sequences or oftheir specific protein products, but also of agonists and theirreceptors. It also makes possible in vitro and in vivo theidentification of viruses, bacteria, parasites or their specificcomponents, as well as pathogenic agents of prion type.

The apparatus of the invention can thus be used:

for purposes of analysis of different types of genomes and notably fortheir sequencing,

for identification of cells, tissues and organs or of different types ofnormal or pathological specific receptors,

for purposes of diagnosis as, for example, thanks to the identificationof genes, their component or their product,

for purposes of screening of molecules or chemical or biologicalsubstances with known or potential therapeutic characteristics,

for follow-up of the activity of new or already known therapeutic agents(possibility of multistage and repeated guided examinations).

The above list is not exhaustive, and with this information the expertis capable of extending the use of the apparatus of the invention to anyother type of analysis, such as those employing the antigen-antibodybond or ligand systems to the extent, at least in part, that the bondsoccurring in vivo are detectable by means of the microsystems described,even if additional bonds are subsequently formed to reveal ex vivo or invitro a reaction having taken place in vivo and in situ.

The apparatus of the invention offers, in particular, a significantimprovement in methods of diagnosis, therapeutic indication, treatmentand therapeutic follow-up, as well as in screening of drugs havingoriginated from genomic and proteinomic (or proteomic) research.

The apparatus of the invention is noteworthy in that it is noninvasiveor microinvasive and nondestructive and makes possible the remoteidentification of all chemical or biological substances capable ofreacting more or less specifically with active substances fixed oninvestigation microsystems. It can involve, notably, molecules orsubstances present exclusively in situ in the organ or tissue or incells (intracellular fusion). This exclusive presence is linked to thefact that these molecules or substances are rapidly metabolized outsidethe tissue studied as soon as they depart from their local environment.This demonstrates the importance of operating in situ with themicrosystem described.

It is therefore suitable for analysis or treatment:

in vitro: on natural or genetically modified cell, tissue or organcultures originating from specimens taken from living beings;

in vivo: under invasive or microinvasive conditions. It makes possiblethe guidance, orientation and positioning of the microsystem intended,notably, for molecular hybridization in situ in target cells, tissues ororgans.

The apparatus of the invention is further particularly noteworthy, forit makes it possible to reach, for analysis or treatment, sitesinaccessible or hard to reach by conventional methods.

The flexible rod makes it possible to bring and then position themicrosystem in contact with the substrate to be analyzed, whetherinvolving a living or expired biological material (fresh, fixed, frozen,mummified or fossilized), consisting of identical or comparable cells,mono- or multicellular tissues or organs, directly or aftermicroeffraction of the mesothelium or protective epithelium and of theconnective capsule as well as vascular endothelia in case ofendovascular exploration. The flexible rod ensures the strength andcohesion of the apparatus and its flexibility and transmissibility ofthe movements imparted by remote control manually or robotically. Theapparatus must, of course, be biocompatible and advantageously sterile,at least in its terminal part in contact with the substrate to betreated or analyzed.

The apparatus of the invention therefore includes, in addition to themicrosystem where the chemical and biological reactions are carried out,a rod making possible the manual or robotic control of the apparatus.

The flexible rod controlling the apparatus makes it possible to ensureat least the following two functions:

Guidance of the microsystem up to the substrate analyzed or treated

Orientation and positioning of the microsystem in the substrate analyzedor treated, including the opening into a flexible catheter which will inturn be initially guided by an endocavitary or endovascular explorationsystem. In some cases the microsystem can be directly entered at thehollow end of the endocavitary or endovascular exploration system.

The flexible rod is advantageously designed to cooperate with differentmedical instruments used for diagnostic, therapeutic or experimentalpurposes. It is a question of apparatuses that are:

noninvasive, intended for the exploration of natural cavities in contactwith the outside environment, like instruments for ORL,bronchopulmonary, digestive, urological or gynecological exploration;

microinvasive, with a view to exploration of natural cavities not indirect contact with the outside environment; it is then a question ofinstruments used in arthroscopy, colonoscopy, etc., or of endovascularexploration systems, or even superficial tissue or organ biopsy tools,notably used transcutaneously, like parenchymal effraction systems, suchas needles, sharp points, cutting devices, etc.

The flexible rod is thus combined with one of those medical instruments.According to a preferred embodiment, the flexible rod is microadapted tobe introduced in the above-mentioned instruments possessing an internalopening. The flexible rod can then slide into that internal opening bymeans possibly of a guiding groove specially provided for that purposeand thus make possible in vivo control of the apparatus of the inventionin the course of different stages of a diagnostic or therapeuticexploration.

But the flexible rod can also consist of those different medicalinstruments themselves, which are then used as rods for control of theinvestigation or treatment microsystem.

The microsystem is attached to one of the ends of the flexible rod,either on the outside of the latter or inserted in the opening of saidflexible rod by any means of connection. That means of connection can bea pivot system making possible the remote-controlled orientation andpositioning of the microsystem in space from the flexible rod. The meansof connection can consist of a deformable material electronicallyremote-controlled. It can also involve a biological glue, notably a typeof glue used for bone tissue repair.

In all cases, the means of connection must satisfy the followingcriteria: adherence, strength and flexibility (resistance to handlingand to stresses associated with remote-controlled movements),biocompatibility and sterility.

The apparatus of the invention can be made from a mold of adaptiveconfiguration into which one or more homogeneous materials or electroniccomponent materials are poured in order to take the desired shape. Themeans intended for making the pivot system and possibly a temporary maskcan be cast at a chosen location of the mold either in the form of anintermediate device or in the form of material suitable for constructingthe entire apparatus described above. It thus gains in efficiency, whileretaining its functional coherence.

The means of connection can be operable by remote control to release themicrosystem in situ. Thus, the apparatus of the invention comprises thetwo embodiments described below.

According to a first embodiment of the apparatus of the invention, thecontrol rod makes it possible to introduce, position and then extractthe microsystem after the chemical and biological reactions sought areproduced in the substrate.

In order to improve contact between the microsystem placed in situ andthe substrate, a rotary or back-and-forth motion can be locally impartedto the microsystem by means of the flexible guide rod, whether thelatter is solid (the microsystem is then attached at its end by anappropriate system, possibly a robotic ball joint), or is hollow andtubular, in order to receive the base of the microsystem which is thenpartially inserted there. The objective is laceration of the tissue, oreven cell lysis, through the introduction of appropriate reactivesubstances, conveyed by the catheter to the microsystem.

These operations are controlled either manually or by roboticassistance. For applications of analysis of a substrate, once recovered,the microsystem is analyzed in the laboratory by standard methods ofrevealing chemical reactions, notably of receptor/ligand type, which areproduced, possibly after preparatory stages like a polymerization chainreaction. In that embodiment the microsystem is released from theflexible rod after extraction and the means of connection does not needto be remote-controlled. The means of connection must, however, ensurecohesion and sufficient flexibility to permit remote control with a viewto guidance, orientation and positioning and possibly the extraction ofthe microsystem in situ.

According to a second embodiment of the apparatus of the invention, themeans of connection between the flexible rod and the microsystem can beremote-controlled to position the apparatus on the site to be analyzed.The microsystem can be transported and positioned by means of acompatible sterile balloon. In that embodiment the microsystem can beanalyzed by remote control thanks to systems of sensors making possibleanalysis of the chemical or biological reactions which are possiblyproduced in the investigation microsystem. Any microprocessor or anyapparatus capable of increasing the sensitivity, efficiency and,therefore, performance of the microsystem or the remote control ofplacement (guidance, orientation and positioning) of the microbiochipcomes within the scope and can be used within the scope of the apparatusaccording to the invention.

The apparatus of the invention can include a system of protection of theinvestigation microsystem, which is removed once the latter is broughtto and positioned on the site to be analyzed. That system of protection,for example, a removable screen or deformable net, can cover the entiremicrosystem or only its active surface. That system of protection can besituated in the means of connection or placed in the microsystem itself.

The apparatus of the invention can include, at the end where themicrosystem is attached, any system of assistance to operation of themicrosystem, like a heating and/or cooling system, system for release ofbiological or chemical substances like buffers, development reagents,any biological alteration product or any molecules of the cellenvironment.

It can be supplemented by any system capable of lacerating the tissueexamined or by a system introducing substances capable of planned lysisof the cells in order to have access to the content of the latter, whileremaining in situ long enough for an optimal fixation reaction of themolecules of the substrate on those of the reactive layer.

The apparatus of the invention can be combined with any means makingpossible by remote control:

monitoring by sensory receptors (tactile, optical, physicochemical and,notably, electronic or computer-digitized) or for any system of signalcapture or processing,

the performance of biopsies, whatever the size,

treatment, for example, of tumors,

local injection of chemical or biological products (cells or tissuescarrying or not carrying gene vectors or components of same, as well asindependent vectors), cell or tissue products, chemical orphysicochemical molecular agents, and labeling agents of any kind,radioactive or not).

The apparatus of the invention can be used concomitantly or successivelywith means based on chemical or biological reactions other than thoseused in the microsystem, or with complementary means of cell laceration,cell lysis, biopsy, injection or sensory capture.

The apparatus of the invention is noteworthy in that it can be used onany type of biological or chemical substrate belonging to beings livingor dead, human, animal or vegetable.

For example, it is useful in the animal, under normal conditions orunder experimental conditions, for pathological studies, or intransgenic animals presenting malignant tumors or different pathologicaldisorders, as well as in the course of cell, tissue or organ grafts inorder to monitor tolerance or rejection. It makes it possible to followthe biological course of animals for the development of selectivetreatments, selection of animal breeds, and monitoring of viral,microbial and parasitic diseases as well as diseases from prion-typeagents. The apparatus of the invention makes it possible to improve, inthe animal, techniques of reproductive cloning or those intended toobtain cell lines with regenerative therapeutic activity originatingfrom embryonal stem cells or from stem cells taken after birth called“adult” stem cells. These are cells from the vessels of the umbilicalcord, but also autologous adult cells activated in vitro and presentingthe characteristics of stems cells capable of being subsequentlydifferentiated in situ, notably after guided regenerative therapy in themyocardium.

In the human being the apparatus of the invention makes it possible,noninvasively or microinvasively, to prepare the isolation andsequencing of genes and/or the identification of their functionalproduct, to specify a diagnosis by chemical or biological methods, toidentify therapeutic indications more pertinently, to test newbiological preparations or new molecules with therapeutic activity, andto follow therapeutic effectiveness thanks to multistage guidedexaminations, repeated without major risk, considering the noninvasiveor microinvasive nature of use of the apparatus. The apparatus of theinvention is thus applicable to the analysis of benign or malignanttumor pathology, involving solid or fluid tumors like cancers orleukemia, and neurological, muscular, hematological, cardiovascular,metabolic or degenerative pathologies, notably whether linked or not toan identified genetic anomaly as well as to a pathologicalpredisposition with a genetic component. The apparatus of the inventionis also suitable for the practice and monitoring of cell therapy, genetherapy, regenerative treatment carried out with embryonal stem cellcultures or stem cells taken after birth or even for preimplantationdiagnosis after in vitro fertilization (IVF). The apparatus of theinvention is also useful in the developing human being, for example inthe embryo after IVF in observance of the ethical rules in effect, inthe fetus within the scope of a prenatal diagnosis with a view, notably,to the diagnosis of genetic anomalies, but also within the scope ofprenatal therapy carried out in utero, in which use of the apparatuscould expand the indications. The apparatus of the invention is alsoapplicable in legal medicine and in the course of forensicinvestigations.

In plants or in the environmental field, the apparatus of the inventioncan be a valuable tool for the identification and selection of plantvarieties, creation of genetically modified organisms, monitoring ofviral or parasitic diseases, soil surveys and monitoring of ecologicalimbalances of a biological or chemical nature, thanks to in vitro and invivo applications, notably for the identification of biological orchemical pollutants of ecosystems.

In the food and agriculture field, the apparatus of the invention isuseful for the development and follow-up of GMOs, notably, formonitoring gene regulation and expression, for monitoring possiblecontamination throughout the food chain without prior denaturation offoodstuffs, for example for the identification of microorganisms,parasites, viruses, rickettsiae or proteins of prion type. Thus, theapparatus of the invention is useful for monitoring different stages ofpreservation processes, notably by freezing.

The apparatus of the invention is also of interest in thepaleontological field for identification and analysis of the chemical orbiological characteristics of mummified or fossilized cells, tissues andorgans, with the advantage of not destroying the objects analyzed, andit makes it possible to improve the tools for identification of thedifferent stages of the evolution of species.

The invention will be better understood by reading the followingexamples concerning one particular embodiment of the apparatus of theinvention, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a working example of an apparatus according to theinvention,

FIGS. 2 and 6 represent a general diagram of immunocapture by the ELISAmethod, with an anti-mannan antibody and with an anti-laminin antibodyrespectively,

FIG. 3 shows a model of a flexible system used for guidance of thesupport,

FIG. 4 represents a diagram of the support presenting a plurality ofregions sensitized by an antibody,

FIG. 5 shows a pilot apparatus in which the rigid plastic support isinserted in the guidance system.

This work was carried out with a system of “in situ” sampling of ananalyte for its subsequent characterization in vitro. The analytesampling was performed by immunocapture or affinity capture and thesampling system presented the following characteristics:

rigidity,

guidance by a flexible system,

compound of a polymer making possible the coupling of the antibody (Ab)or of the ligand for an immunocapture or affinity capture of theanalyte.

Two models were adopted for this work:

A first model entails the analysis of a disseminated candidiasis in themouse upon disclosing antigens (Ag) of Candida albicans mannans type inthe kidneys or liver.

A second model is based on analysis of the Engelbreth-Holm-Swarm(E.H.S.) tumor in the mouse upon disclosing laminin in the tumor of thethigh.

For study of these two models, an immunocapture system was used with theaid of anti-Candida Ab or anti-laminin Ab.

The different parameters mentioned above were studied in advance in amicroplate system:

Ab concentration for immunocapture

Ab specificity for immunocapture

Biotin labeling of the revealing Ab

Detection of the biotinylated Ag by the streptavidin-enzyme complex

The following characteristics of the supports were studied in an invitro immunocapture system:

their nature: polystyrene or other

activation by different agents

Ab coupling on the supports

The results obtained on this work show that it is possible (i) to fix ona rigid support (joined to a flexible rod) two antibodies of differentspecificities (anti-Candida and anti-laminin), (ii) to samplecorresponding analytes (Candida and laminin antigens), and (iii) toidentify those analytes by antibodies labeled with biotin which is thenrevealed by a streptavidin-enzyme complex.

I—Conception and Development of the System for Disclosing C. albicansMannan Antigens

1) Principle of the reaction

It involves an immunocapture by the ELISA method on microplate or onplastic supports (ELISA: Enzyme Linked Immuno Sorbent Assay), theprinciple of which is represented on the attached FIG. 2.

2) Preparation and control of the reagents

a) Purification of the Anti-mannan Monoclonal Antibody (Anti-M Ab)

The anti-mannan monoclonal antibody was purified and supplied by SR2B.

b) Labeling of the Anti-M with Biotin

The purified antibody, at 3 mg/ml, is dialyzed overnight at 4° C. in thepresence of borate buffer 0.1 M pH 8.8. A biotin solution (ester of6-biotinamidocaproylamidocaproic acid and N-hydroxysuccinimide; Sigma®)at 10 mg/ml in DMSO is then added at the rate of 50 μg/mg of antibody.After 4 h of incubation at room temperature and stirring, ammoniumchloride 1 M is added at the rate of 20 μl/250 μg of biotin, and thesolution obtained is incubated again for 10 min. at room temperature.

After stopping of the reaction, the labeled antibody is precipitated byammonium sulfate 2M and, after centrifugation, the base is taken up by aTBS buffer (tris-HCL 20 mM, pH 7.5, NaCl 150 mM) and dialyzed for 24hours at +4° C. in the presence of that same buffer.

This labeled antibody is kept in aliquot form at −20° C.

c) Preparation of the Mannan Antigen

The different stages of preparation are as follows:

Obtaining blastospores of Candida albicans ATCC 66396, 48 hours onSabouraud Dextrose Agar at 22° C.

Lyophilization of yeasts

The lyophilisate is taken up by a citrate buffer pH 7.2, 0.2 M

Autoclaving for 2 hours at 131° C. (1.3 bar)

Centrifugation of the suspension for 15 minutes at 3000 rpm

Addition of 2 volumes of absolute ethanol to the supernatant andprecipitation overnight at +4° C.

Centrifugation for 15 minutes at 3000 rpm and washing of the base with60% ethanol;

Centrifugation for 15 minutes at 3000 rpm and addition of acetone todehydrate the base

Take-up of base in distilled water

The carbohydrate determination is made by the Dubois method. Thecarbohydrate concentration obtained is 21 mg/ml.

d) Choice of Plastic Supports

Flexible systems

Among the different flexible systems tested for guidance of the support,the one represented on the photographs given in the attached FIGS. 3 to5 was adopted. It involves a plastic flexible hollow tube in which thesupport is fitted.

Rigid plastic supports

Three types of plastic supports were chosen and used for development ofthe system, one described as “yellow” plastic, one described as “blue”plastic and one described as “white” plastic. For each, three methods ofcoupling were tested, as represented on the attached FIG. 4: couplingwithout treatment (ø), coupling with treatment by coupling agent 1 (A1),and coupling with treatment by coupling agent 2 (A2). The three types ofsupport were tested untreated or treated with A1 or A2 and for eachsystem the supports were used sensitized with the anti-M Ab (U). The“white” and “blue” supports gave similar results. Only the supportconsisting of “white” plastic, 2 cm long and treated with coupling agentA2, was adopted for continuation of the experiments.

e) Control of Reagents and Determination of the Parameters ofSensitivity and Specificity on Microplate and on Plastic Supports invitro

On microplate

The method was applied on microplate for ELISA (Greiner®). Theparameters adopted for a maximum sensitivity are: concentration of theanti-M Ab for immunocapture at 1 μg/ml, saturation in 10% PBS-milkovernight at +4° C., and concentration of biotinylated anti-M Ab at 0.01mg/ml. With those parameters the sensitivity is 0.01 μg/ml for thecarbohydrate (and not mannan) concentration.

On plastic supports

The method was applied on hemolysis microtubes (Fisher®). The parametersadopted for a maximum sensitivity are: concentration of the anti-M Abfor immunocapture at 10 μg/ml, saturation in 10% PBS-milk overnight at+4° C., and concentration of biotinylated anti-M Ab at 0.02 mg/ml. Withthose parameters the sensitivity is 0.01 μg/ml for the carbohydrate (andnot mannan) concentration.

3) Dislosure of mannan Ag ex vivo

a) Determination of Parameters for Obtaining a Disseminated Candidiasisin the Mouse

Use of Candida albicans strain ATCC 66396, culture on Sabouraud DextroseAgar for 24 hours at 37° C.

Initial yeast concentration 10⁷/ml in NaCl 0.15M.

Inoculation of 100 μl IV in the caudal vein of the mouse.

Obtaining a disseminated candidiasis with presence of renal and hepaticabscesses.

b) Disclosure of Mannan Ag in the Liver, Kidneys and Blood

On day D=0: intravenous inoculation in mice of blastospores of C.albicans ATCC 66396.

On D=2, the mice are sacrificed.

The blood is collected by periorbital sampling in glass tubes and afterdecantation the serum is tested for mannan Ag in the bloodstream.

The kidneys or liver are sampled and placed in a sterile Petri dish. Theimmunocapture is carried out by placing the “white” plastic supportsensitized with the anti-M Ab in those organs.

After an incubation of 15 minutes, the search for mannan Ag on thesupports is made according to the principle described in vitro.

The kidneys, liver or blood of healthy mice constitute the negativecontrols.

c) Results

The results are expressed in optical density (OD) after subtraction ofthe OD of the controls, which are the healthy mice.

Concerning the kidneys, the right kidney and the left kidney of micesuffering from candidiasis were tested independently from the kidneys ofhealthy mice. A significant difference was observed between the signalobtained for the presence of mannan Ag in the “sick” kidneys and thesignal obtained for kidneys of healthy mice (for example, the opticaldensity difference is approximately 0.4).

Concerning the liver, the mannan Ag were also detected by the probe witha significant difference (optical density difference 0.6).

In the blood (diluted to 1/10) of healthy mice or mice suffering fromcandidiasis, no mannan Ag were detected in the bloodstream.

II—Conception and Development of the System for Disclosure of Laminin

1) Principle of the reaction

It involves an immunocapture by ELISA method on microplate or on plasticsupports (ELISA: Enzyme Linked Immuno Sorbent Assay), the principle ofwhich is represented on the attached FIG. 6.

2) Preparation and control of reagents

a) Labeling of the Anti-laminin Antibody (Anti-L Ab) with Biotin

The antibody adopted for developing the detection of laminin is apolyclonal antibody produced in the rabbit and purified by affinity(Rockland®). The method of labeling with biotin is identical to thatused for the anti-M.

b) Laminin

The laminin adopted for in vitro development is the purified lamininfrom mice (Sigma®).

c) Choice of Plastic Supports

Flexible systems, as previously described for Candida albicans.

Rigid plastic supports, as previously described for Candida albicans.

d) Control of Reagents and Determination of the Parameters ofSensitivity and Specificity on Microplate and on Plastic Supports invitro

On Microplate

The method was applied on microplate for ELISA (Greiner®). Theparameters adopted for a maximum sensitivity are:

Concentration of the anti-L Ab for immunocapture at 5 μg/ml.

Saturation in 10% PBS-milk overnight at +4° C.

Concentration of biotinylated anti-M Ab at 5 μg/ml.

With those parameters, the sensitivity is 0.01 mg/ml for the lamininconcentration.

On Plastic Supports

The method was applied on hemolysis microtubes (Fisher®). The parametersadopted for a maximum sensitivity are the same as on microplate, namely:

Concentration of the anti-L Ab for immunocapture at 5 μg/ml.

Saturation in 10% PBS-milk overnight at +4° C.

Concentration of biotinylated anti-L Ab at 5 μg/ml.

With those parameters, the sensitivity is 0.01 mg/ml for the lamininconcentration.

4) Disclosure of laminin ex vivo

a) Determination of Parameters for Obtaining a “Laminin” Tumor Visibleto the Naked Eye

Thawing of the E.H.S. (Engelbreth-Holm-Swarm) sarcoma in the mouse.

Inoculation in the thighs of mice.

After three weeks the mice are sacrificed, and tumor samples are taken,ground up and inoculated in other mice.

b) Disclosure of Laminin in the Tumor of the Thigh in the Mouse

The mice are sacrificed and immunocapture is carried out by placing the“white” plastic support sensitized with anti-L in the tumor of thethigh. Thighs of healthy mice constitute the negative controls.

c) Results

The results are expressed in optical density (OD) after subtraction ofthe OD of the controls, which are the healthy mice.

A significant difference is observed between the signal obtained for thepresence of laminin in the tumor and the signal obtained for thighs ofhealthy mice (for example, the optical density difference isapproximately 0.3).

III—Coupling of the Two Systems: Simultaneous Detection of Mannan Ag andLaminin ex vivo

1) Sensitization of the probe

The support constituted by the “white” plastic and maintained by theflexible guidance system is sensitized with the anti-M Ab and anti-L Abat the previously established concentrations.

2) Disclosure of mannan Ag and laminin ex vivo

The immunocapture is carried out in:

healthy mice (controls)

mice suffering from candidiasis

mice with a laminin tumor (E.H.S.).

Supports are:

placed first in the kidneys or liver of a mouse suffering fromdisseminated candidiasis and then in the tumor (E.H.S.) of the thigh ofanother mouse,

or placed first in the tumor (E.H.S.) of the thigh of one mouse and thenin the kidneys or liver of another mouse suffering from disseminatedcandidiasis.

The supports are then revealed by the anti-M or anti-L or a mixture ofboth.

Negative controls are made by placing supports in the kidneys or liverof a healthy mouse and then in the thigh of the same mouse (and viceversa).

3) Results

The results are expressed in optical density (OD) after subtraction ofthe OD of the controls, which are the healthy mice.

Whatever the order in which the support is implanted in the differentorgans, the mannan Ag or the laminin is significantly detected inrelation to the healthy mice, when the system is revealed by only one ofthe two antibodies (OD: 0.4 for the mannan Ag and 0.4 for the laminin).

When the mixture of both biotinylated antibodies is used for revealing,the signal obtained is much higher (for example, mouse withcandidiasis+laminin OD=0.7).

The mannan Ag could not be detected in the blood of the healthy mice orof the mice suffering from disseminated candidiasis.

What is claimed is:
 1. An apparatus comprising a support which iscapable of non-fluorescent biological assaying or active agent delivery;a flexible rod; a system for protecting the support, and a systemcapable of lacerating a tissue, wherein one end of the flexible rod isattached to the support and the other end of the flexible rod isattached to a device, which controls the movement of said support, andwherein the flexible rod is encased in an internal opening of adiagnostic, therapeutic or experimental medical instrument.
 2. Theapparatus of claim 1, wherein the support is capable of non-fluorescentbiological assaying.
 3. The apparatus of claim 2, wherein the supportcomprises an array of chemical or biological substances.
 4. Theapparatus of claim 3, wherein the support comprises an array of at least2 chemical or biological substances.
 5. The apparatus of claim 3,wherein the support comprises an array of at least 100 chemical orbiological substances.
 6. The apparatus of claim 3, wherein the supportcomprises an array of at least 1000 chemical or biological substances.7. The apparatus of claim 3, wherein the chemical or biologicalsubstances are capable of reacting with one or more target substancespresent in a substrate.
 8. The apparatus of claim 3, wherein thechemical or biological substances are one or more substances selectedfrom the group consisting of a receptor, a ligand, an antigen, anantibody, a peptide, a polypeptide, a protein, and a polynucleotide. 9.The apparatus of claim 8, wherein the chemical or biological substancesare polynucleotides.
 10. The apparatus of claim 8, wherein the chemicalor biological substances are one or more of a peptide, a polypeptide ora protein.
 11. The apparatus of claim 1, wherein the end of the flexiblerod attached to the support further comprises at least one pivotattached to the support.
 12. The apparatus of claim 1, wherein thesupport is in the form of a ribbon and is wound around the end of theflexible rod.
 13. The apparatus of claim 1, which further comprises aheater or cooler positioned at the end of the substrate.
 14. Theapparatus of claim 1, wherein the support is capable of active agentdelivery.
 15. The apparatus of claim 14, wherein the active agent is apharmaceutically active agent.
 16. The apparatus of claim 14, whereinthe active agent is at least one agent selected from the groupconsisting of a nucleic acid, a protein, and an antibody.
 17. Theapparatus of claim 1, which further comprises a biopsy device.
 18. Theapparatus of claim 1, wherein the system of protecting the support isthe internal opening of the diagnostic, therapeutic, or experimentalmedical instrument.
 19. The apparatus of claim 1, wherein the flexiblerod is capable of sliding in the internal opening of the diagnostic,therapeutic, or experimental medical instrument.
 20. A method ofassaying a biological sample, comprising contacting the biologicalsample with the support of the apparatus of claim 3 to assay thebiological sample.
 21. The method of claim 20, wherein the biological orchemical substances are one or more substances selected from the groupconsisting of a receptor, a ligand, an antigen, an antibody, a peptide,a polypeptide, a protein, and a polynucleotide.
 22. The method of claim21, wherein the chemical or biological substances are polynucleotides.23. The method of claim 21, wherein the chemical or biologicalsubstances are one or more of a peptide, a polypeptide or a protein. 24.The method of claim 20, wherein the support comprises an array of atleast 2 chemical or biological substances.
 25. The method of claim 20,wherein the support comprises an array of at least 100 chemical orbiological substances.
 26. The method of claim 20, wherein the supportcomprises an array of at least 1000 chemical or biological substances.27. The method of claim 20, wherein the biological sample is a naturalcell, genetically modified cell, tissue culture, or organ culture. 28.The method of claim 20, wherein the biological sample is an in vivocell, tissue, or organ.
 29. A method of delivering an active agent to alocation in a patient, comprising inserting the apparatus of claim 14 tothe location in the patient, and delivering the active agent.
 30. Themethod of claim 29, wherein the active agent is a pharmaceuticallyactive agent.
 31. The method of claim 29, wherein the active agent is atleast one agent selected from the group consisting of a nucleic acid, aprotein, and an antibody.